Versa 8

CD8 immunofluorescence analysis of FFPE samples was performed with CD8A antibody (SP57, Ventana 790-4460) in a College of American Pathologist (CAP)-controlled area within the Oncology and Immunology Unit of WuXi AppTec using the IF 6-colorWJJ-CD30 protocol on a Leica BOND Rx platform. Whole-slide images were acquired by Leica Aperio VERSA 8. Z1. Image analysis was performed using the HALO software package (Indica Labs, United States).

Tissue microarray slides with cores containing mouse normal mammary gland and tumor sections were stained for Panel A and Panel B markers for multiplex IF and scanned with a Leica VERSA 8 whole-slide fluorescent digital scanner as described above. IF was quantified with the image analysis tool in ImageScope v 12.4.3, and a cellular IF algorithm was selected and modified for each panel. Four different algorithms were tuned for each panel and tissue type. First, the cellular IF algorithm was tuned to segment cellular nuclei to ensure accurate identification and quantification of 4′,6-diamidino-2-phenylindole (DAPI) -stained nuclei. Next, the algorithm was tuned for positive fluorescence intensity to quantify the tissue expression of each marker while minimizing non-specific background staining or autofluorescence. Once the tuning was completed for the individual markers, co-expression classes of selected markers within cells were created and included in the algorithm quantification. To evaluate the lymphatic vessel and mammary duct cellular environment, each vessel and duct were individually manually annotated in the images of the mammary gland cores for structural and functional analysis with these algorithms. Ductal expression was quantified as positive cells per annotated duct structure and expression per duct for each duct was plotted from all mice as a group.

The experiments were performed as previously described (An et al., 2022 ; Wang et al., 2020 ; Yang et al., 2022 ). The paraffin sections of colon tissue underwent gradient dehydration and were treated with 1 mM EDTA (pH 8.0) in a microwave oven for 30 min for antigenic repair. The sections were incubated with primary antibodies overnight at 4 °C followed by a General purpose SP Kit (ZSGB-Bio) and DAB immunochromogenic reagent (Gene Tech) according to the manufacturer's instructions. The sections were then stained with hematoxylin (Beyotime Biotech). Signals were imaged with an Aperio VERSA 8 (Leica) multifunctional scanner. Primary antibodies used were anti-OTUD4 (Abcam, ab106368), anti-lysozyme (Abcam, ab108508), and anti-Ki67 (CST, 12202S).

Immunohistochemistry (IHC) was done by WuxiAppTec, China. OCT blocks of tumor samples were sectioned (4 µm thickness/section), and sections on slides then were warmed at room temperature for 30 min, fixed in ice cold 70% ethanol for 15 min and air dried for 15 min before being processed using Leica EG Bond RX. Antibodies used are listed in Supplementary Table S1. All stained slides were scanned with Leica Aperio VERSA 8, and images were analyzed using the HALO™ image analysis platform. Areas of necrosis were excluded. Total cell numbers (or area) and IHC positive cells (or area) were scored. The IHC score represents the positive to total cell count/area ratio in the examined section.

IF staining was performed by utilizing AF488-labeled ssDNA T9 aptamer against ManLAM (Tang et al., 2016 (link)). Sectioned specimens (5~7 μm) were incubated at 60°C for 1 h, then deparaffinized and rehydrated with consecutive dilutions of alcohol (absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol). After antigen retrieval, samples were blocked with 100 μl 5% BSA at room temperature for 30 min, then 100 μl 300 nM AF488-labeled ssDNA aptamers or AF488 labeled anti rabbit-IgG against rabbit anti-Rv2645 (1:300) and anti-Rv1579 polyclonal antibodies (1:500) were used to detect the M.tb antigens in the lung sections from M.tb infected mouse were added and incubated at 37°C for 1 h. Lastly, sections were sealed with Anti-fade Fluorescence Mounting Medium. Images were analyzed by using a FISH multi-function scanner (Aperio VERSA 8, Leica) or using a normal fluorescence microscope.

Formalin-fixed, 4T1-mCD19 tumors were embedded in paraffin, sectioned, and stained with anti-mouse CD3 antibody in a blinded manner by a histology technologist. Images were obtained using a Leica Versa 8 whole-slide scanner.

Digital images of stained slides were acquired using Leica's Aperio VERSA 8 automated microscope. The invasive margin (IM) and the center of the tumor (CT) were defined in each digital image. IM was defined as the area expanding 300 μm into the stroma and 50 μm into the tumor measured from the stromal tumor borderline. CT was defined as an area remote from the stromal tumor borderline in the depth of the tumor bulk. All areas matching these criteria were included in the analysis of each slide, except from areas with obvious staining artefacts or damaged tissue that were excluded from further analysis. Image analysis of the IM and CT areas was performed using ImageScope software package (Leica Microsystems, Wetzlar, Germany). The number of stained cells and the size of the measured region were recorded in each area of IM and CT, and the density of stained cells (number of cells per square mm) was calculated from these data.